Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine and plays a critical role in diverse cellular events, including cell proliferation, differentiation, apoptosis and necrosis. Through binding to its two transmembrane receptors, TNF-R1 (p55) and TNR-R2 (p75), TNF is a major mediator of both inflammation and immunity, and has thus been implicated in a variety of pathological inflammatory conditions and autoimmune diseases. Since the discovery of its tumoricidal activity, the TNF pathway has become one of the most studied of signaling pathways, resulting in the characterization of a vast superfamily of receptors, known as the TNF receptor superfamily, and their ligands. In response to TNF treatment, the transcription factor NF-kappaB and MAP kinases, such as c-Jun N-terminal kinase (JNK), are activated in most types of cells and, in some cases, apoptosis or necrosis can also be induced. Much is known about the molecular mechanisms of TNF signaling. Engagement of TNF-R1 by the TNF homotrimer initiates the binding of the adaptor protein, TRADD (TNF receptor 1-associated death domain protein) which then recruits other effector proteins, such as RIP1 (receptor interacting protein) and TRAF2 (TNFR-associated factor 2) to form a TNF-R1 signaling complex leading to the activation of several pathways, including NF-kappaB and MAP kinases. Both TRAF2 and RIP1 play essential roles in the activation of NF-kappaB and MAP kinase pathways through recruitment and activation of IKK (IkB kinase) and MAP3Ks to the complex. Under certain conditions, it is thought that the complex of TRADD, RIP1, and TRAF2 proteins dissociates from the receptor and forms a secondary protein complex containing other proteins. Apoptosis is primarily initiated through the recruitment of the death domain protein FADD (Fas-associated death domain protein) to this secondary complex. FADD recruits and induces dimerization and activation of the autocatalytic activation of the initiator cysteine proteases, caspases-8 and -10, which drive apoptosis. Although caspase cleavage is the primary means of initiating apoptotic cell death by TNF, cell death still occurs (or is even enhanced) in some types of cells in the absence of caspase activity. While the mechanism of TNF-induced apoptotic cell death is well elucidated, the signaling events that lead to TNF-initiated caspase-independent death are largely unknown. Caspase-independent necrotic cell death has been proposed to involve reactive oxygen species (ROS) that have been suggested to be derived from the mitochondria. ROS can inactivate MPK phosphatases, leading to sustained JNK activation and resulting in eventual cell death. RIP1 is necessary for the generation of ROS by TNF and is required for the initiation of necrotic cell death. During apoptotic cell death, RIP1 is cleaved by caspase-8, limiting its ability to activate the pathway(s) of ROS generation. NADPH oxidases are enzymes specifically dedicated to the production of ROS. TNF is known to stimulate the activity of one such oxidase (Nox2/gp91phox) in macrophages and neutrophils, resulting in the generation of superoxide (O2-) that is important in the ability of these cells to kill invasive microorganisms. Nox2, which is a membrane glycoprotein that exists as a heterodimer with a 22-kDa subunit (p22phox), is activated by the p47phox and p67phox proteins and mutations in any of the four oxidase subunits can result in chronic granulomatous disease (CGD), characterized by a susceptibility to severe and recurrent bacterial and fungal infections derived from the inability of phagocytic cells to destroy pathogens. When phosphorylated, the p47phox subunit binds to membrane phospholipids, interacts with p22phox, and recruits the p67phox subunit to the complex. The p67phox activator binds and stabilizes an interaction of the complex with the small GTPase Rac, and the fully formed complex is able to generate O2- in the presence of NADPH. TNF is a potent activator of Nox2. However, the mechanism of activation is unclear, but may involve phosphorylation of p47phox. NADPH oxidases have been characterized in non-phagocytic cell types, along with novel regulatory adaptor proteins. Most require the presence of the small subunit p22phox for activity. The regulatory p41NOXO1 and p51NOXA1 subunits may function in some of these oxidase complexes similarly to p47phox and p67phox, respectively. Unlike p47phox, NOXO1 lacks an autoinhibitory region, binds to different lipids, and does not require phosphorylation for membrane translocation. Nox1 and Nox4 appear to be more ubiquitous than other NADPH oxidases in non-phagocytic cells. However, Nox4 is constitutively active when complexed with p22phox and does not appear to require other additional regulatory subunits and may be regulated solely by direct transcriptional control of its expression level. Therefore, the remaining oxidase, Nox1, may be the most likely source of immediate NADPH oxidase activity in response to TNF in non-phagocytic cell types where Nox2 is not expressed. Recently we showed that the Nox1 NADPH oxidase is activated during TNF-induced necrotic cell death through forming a complex with TRADD, RIP1 and Rac1. Interactions of NOXO1 with TRADD and RIP1 are critical for the activation of Nox1 by TNF and RIP1 is essential for recruiting Nox1 to the complex in MEF cells when necrosis is triggered. Importantly, Nox1-mediated production of O2- is critical for TNF-induced necrotic cell death since such death is inhibited by ROS scavengers, down regulation of the Nox1 protein by siRNA, or expression of dominant-negative versions of TRADD or Rac1. Our data also suggest that Nox1 may mediate the induction of necrosis through regulating sustained JNK activation.